Method for the in vitro synthesis of short double stranded RNAs

ABSTRACT

The present invention relates to the field of synthesis of short double-stranded RNAs. An in vitro transcription method using bacteriophage polymerases and target sequence-specific single-stranded DNA oligonucleotides as templates is disclosed. The present invention finds particularly advantageous use in the synthesis of short interfering RNAs (siRNAs) that have been shown to function as key intermediates in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates and vertebrate systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from PCT/EP02/12165, filed on Oct. 30,2002, which claims priority from a Provisional Patent application No.60/337,975, filed on Nov. 5, 2001. The complete disclosures of theabove-identified applications are incorporated herein by reference intheir entirety.

The present invention relates to the field of synthesis of shortdouble-stranded target-specific RNAs. An in vitro transcription methodusing RNA polymerases and target sequence-specific DNA oligonucleotidesas templates is disclosed. The present invention finds particularlyadvantageous use in the synthesis of short interfering RNAs (siRNAs)that have been shown to function as key intermediates in triggeringsequence-specific RNA degradation during posttranscriptional genesilencing in plants and RNA interference in invertebrates and vertebratesystems.

BACKGROUND OF THE INVENTION

RNA silencing is a remarkable type of gene regulation based onsequence-specific targeting and degradation of RNA. RNA silencing wasfirst discovered in transgenic plants, where it was termed cosuppressionor posttranscriptional gene silencing (PTGS). Only recently asequence-specific RNA degradation process, RNA interference (RNAi),related to PTGS has been found in ciliates, fungi and a variety ofanimals from C. elegans to mice and human cells. Although they maydiffer in detail, RNAi and PTGS result from the same highly conservedmechanism, indicating an ancient origin. The basic process involves adouble stranded RNA (dsRNA) that is cleaved into small double strandedinterfering RNAs (siRNA) which guide recognition and targeted cleavageof homologous mRNA. These small dsRNAs resemble breakdown products of anRNase III-like digestion. In particular, siRNAs are target-specificshort double stranded RNAs wherein each strand of the siRNAs carries5′monophosphate, 3′hydroxyl termini and 3′ overhangs of 2-3 nucleotides(Caplen, N. et al., 2001, PNAS (98) 9742-9747).

RNAi has attracted considerable attention because it is a means ofknocking out the activity of specific genes, being particularly usefulin species that were previously considered not to be amendable togenetic analysis. Recent studies demonstrated that synthetic siRNAs caninduce gene-specific inhibition of expression in C. elegans and in celllines from humans and mice (Caplen N., et al., 2001, PNAS (98)9742-9747; Elbashir S., et al., 2001, Nature (411) 494-498). In saidpublications it was further demonstrated that in mammalian cells siRNAsprovide a sequence specific answer compared to the use of longer dsRNAswhich inactivate the translation factor eIF2α, leading to a generalizedsuppression of protein synthesis. Also, in comparison to inhibition ofgene expression using antisense technology, siRNAs seem to be verystable and thus may not require the extensive chemical modificationsthat single stranded RNA antisense oligonucleotides require to enhancethe in vivo half life.

It is therefore to be expected that RNA silencing using siRNAs willbecome an important tool in engineering control of gene expression aswell as in functional genomics and a variety of biotechnologyapplications ranging from molecular farming to possibly even genetherapy in animals. As different siRNAs may work with differenteffectiveness on their targets, the testing of more than one siRNA for aparticular target will be desirable. In addition, genome-scale reversegenetics programs will require large numbers of siRNAs.

However, production of double stranded target-specific RNA oligos bytraditional chemical synthesis remains relatively slow and expensivewhen compared to DNA oligo synthesis. In addition, chemical synthesis ofRNA oligos requires special synthesizers and complex purificationprotocols. The present invention provides an alternative approach toproduce short double stranded target-specific RNAs based on in vitrotranscription using bacteriophage or other viral polymerases and targetsequence-specific oligonucleotide templates. Compared to the chemicalsynthesis of RNA oligos the present invention is relatively quick andeasy to perform.

However, the in vitro transcribed siRNAs differ from the chemicallysynthesized RNA oligos in two ways. Primarily, identical to the naturaloccurring siRNAs, the chemically synthesized RNA oligos have a5′monophosphate group. The in vitro transcribed siRNAs retain a5′triphosphate group. It was unknown whether the presence of thistriphosphate group renders the in vitro transcribed siRNAs incompetentto induce RNA interference.

Secondly, chemically synthesized RNA oligos are highly purified usingamongst others Ion Exchange and Reverse Phase HPLC wherein purity andquality of the synthesized compounds is further evaluated using amongstothers NMR and mass spectrometry analysis. In the present invention asimple, crude purification protocol is used comprising size exclusionchromatography, phenol:chloroform extraction and ethanol precipitation.It was again uncertain whether the ommitance of an extensivepurification protocol would affect the usefulness of “in vitro”transcribed RNAs in RNA-mediated silencing.

Surprisingly, the present invention demonstrates that the 5′triphosphategroup and the crude purification does not affect the RNA silencingactivity of “in vitro” transcribed RNAs and provides an alternativeapproach to siRNA synthesis which makes it accessible as a research toolin an average molecular biology laboratory.

Existing in vitro methods to synthesize small single stranded RNAs ofdefined length and sequence (Milligan F. et al., 1987, Nucleic Acid Res.(15) 8783-8798), were not directly applicable for the synthesis of smallinterfering RNAs. The problem resides in the fact that RNA polymerasestend to transcribe some nucleotides from the promoter sequence into thetranscript. As a consequence, the target-specific dsRNAs which can beproduced by annealing complementary single stranded RNA moleculesgenerated using the aforementioned methods, must comprise at the 5′endthe nucleotides transcribed from the promoter sequence and at the 3′endthe nucleotides complementary to the nucleotides transcribed from thepromoter sequence. It may well be that in the mRNA of the targetsequence no stretch of a defined sequence length exists wherein the5′-end consists of the nucleotides transcribed from the promotersequence and the 3′-end of the nucleotides complementary to thenucleotides transcribed from the promoter sequence. The presentinvention solves this problem by providing truncated RNA polymerasepromoter sequences wherein one or more nucleotides at the 5′end of thetemplate strand of the promoter sequence are replaced by nucleotidesthat are part of the target-specific sequence. These substitutions donot affect the in vitro transcription yields, but increase thepossibility that at least one target-specific sequence of a definedsequence length exists in the mRNA of the target protein, wherein the5′-end consists of the nucleotides transcribed from the promotersequence and the 3′-end of the nucleotides complementary to thenucleotides transcribed from the promoter sequence.

This and other aspects of the invention will be described herein below.

SUMMARY OF THE INVENTION

The present invention provides an in vitro method for the synthesis ofshort double stranded target-specific RNAs comprising the steps of a)combining a sense target-specific oligonucleotide template and a chainextending enzyme in a reaction mixture such that the template extendedsense oligoribonucleotide product is formed; b) combining an antisensetarget-specific oligonucleotide template and a chain extending enzyme ina reaction mixture such that the template extended antisenseoligoribonucleotide product is formed; and c) hybridizing the senseoligoribonucleotide product obtained in step a) with the complementaryantisense oligoribonucleotide product obtained in step b).

In a further embodiment of the present invention the chain extendingenzyme is an RNA polymerase and the oligonucleotide templates of step a)and b) comprise an RNA polymerase promoter sequence, preferablyconsisting of dsDNA. In a more preferred embodiment the RNA polymeraseis T7 polymerase and the oligonucleotide templates of step a) and b)comprise a T7 RNA polymerase promoter sequence extended at the 5′end ofthe template strand with the target-specific template sequence,optionally extended with 2 or 3 additional nucleotides. The presentinvention finds particular use in the synthesis of small interferingRNAs. It is therefore, a further objective of the present invention toprovide a method for the synthesis of target-specific short doublestranded RNAs, wherein said target-specific short double stranded RNAsare less than 50 nucleotides, preferably less than 30 nucleotides long,even more preferably 30-12 nucleotides long, further characterised bycomprising at the 5′-end nucleotides transcribed from the promotersequence and at the 3′-end nucleotides complementary to the nucleotidestranscribed from the promoter sequence.

Accordingly, the present invention provides a method for the synthesisof small interfering RNAs comprising the steps of a) combining a sensesiRNA template with a chain extending enzyme in a reaction mixture suchthat the template extended sense oligoribonucleotide product is formed;b) combining an antisense siRNA template with a chain extending enzymein a reaction mixture such that the template extended antisenseoligoribonucleotide product is formed; and c) hybridizing the senseoligoribonucleotide product obtained in step a) with the antisenseoligoribonucleotide product obtained in step b); wherein the siRNAtemplates of step a) and b) comprise a double stranded RNA polymerasepromoter sequence extended at the 5′end of the template strand with thetarget-specific template sequence and 2 or 3 additional nucleotides. Ina preferred embodiment the chain extending enzyme is T7 RNA polymeraseand the siRNA templates comprise a double stranded T7 RNA polymerasepromoter sequence, preferably the truncated T7 RNA polymerase promotersequence shown in FIG. 1.

It is a further object of the present invention to provide kits toperform the methods according to the invention as well as the compoundsfor use in any of the methods disclosed.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Oligonucleotide production scheme. An example is given for thedesign of siRNA oligonucleotide templates for a target sequence of 19nucleotides within the coding sequence of JNK2α1 mRNA. Figure disclosesSEQ ID NOS: 1, 19-20 and 2-5, respectively, in order of appearance. TheT7 promoter sequence is disclosed as SEQ ID NO: 56.

FIG. 2A: GL3 target-specific double stranded siRNAs used in a luciferasereporter assay. GL3 siRNA 1, GL3 siRNA 2 and GL3 siRNA 3 were made usingthe in vitro method of the invention. GL3 siRNA oligo was chemicallysynthesized (Vargeese, C. et al., 1988, Nucleic Acid Res. (26),1046-1050) Figure discloses SEQ ID NOS: 21-28, respectively, in order ofappearance.

FIG. 2B: Effects of GL3 target-specific siRNAs and of GL3 antisensesingle stranded siRNAs on luciferase expression in HeLa cells. Cellstransfected with GL3-control luciferase+reporter constructs were takenas 100%.

FIG. 2C: Dose response curve of the GL3-siRNA 1 inhibitory effect onluciferase expression in pGL3-control transfected HeLa cells.

FIG. 3A: EGFP target-specific double stranded siRNAs used in a FACSanalysis of EGFP-transfected HeLa cells. EGFP ds siRNA 2 was made usingthe in vitro method of the invention. EGFP ds siRNA oligo was chemicallysynthesized (Vargeese, C. et al., 1988, Nucleic Acid Res. (26),1046-1050) Figure discloses SEQ ID NOS: 29-32, respectively, in order ofappearance.

FIG. 3B: Effects of EGFP target-specific siRNAs and of EGFP antisensesingle stranded siRNAs on GFP fluorescence in EGFP-transfected HeLacells using FACScan analysis (Beckton-Dickinson). Cells transfected withEGFP DNA only were taken as 100%.

FIG. 4A: JNK2α1 target-specific double stranded siRNAs used in JNK2α1transfected HeLa cells. Figure discloses SEQ ID NOS: 33-38,respectively, in order of appearance.

FIG. 4B: CDS-1 target-specific double stranded siRNAs used in CDS-1transfected HeLa cells. Figure discloses SEQ ID NOS: 39-46,respectively, in order of appearance.

DETAILED DESCRIPTION

This invention relates to the field of synthesis of shortdouble-stranded target-specific RNAs and is based on the in vitrotranscription of oligonucleotide templates using chain extendingenzymes.

Target-specific short double stranded RNAs as used herein refers to adouble-stranded RNA that matches part of the sequence encoding for aspecific protein, i.e. the target protein. These sequences arepreferably less than 50 nucleotides, more preferably less than 30nucleotides long, even more preferably 15-25 nucleotides long. In aparticular embodiment the target-specific short double stranded RNAs areuseful in RNA interference in invertebrate and invertebrate systems assmall interfering RNAs (siRNAs). siRNAs as used herein are short dsRNAmolecules of 12-30 nucleotides, with 2- or 3-nucleotide overhanging3′-ends. In a preferred embodiment the siRNAs are 15-25 nucleotides longwith 2 nucleotide overhanging 3′ends. Even more preferred the siRNAs are17-22 nucleotides long with 2 nucleotide overhanging 3′ends.

In order to obtain dsRNA both a sense and an antisense oligonucleotidetemplate are required. The term “oligonucleotide templates” as usedherein refers to structures that in some direct physical process cancause the patterning of a second structure, usually complementary to itin some sense. In current biology almost exclusively used to refer to anucleotide sequence that directs the synthesis of a sequencecomplementary to it by the rules of Watson-Crick base-pairing (TheDictionary of Cell and Molecular Biology, 3d. Edition, Academic Press,London, 1999 (ISBN 0-12-432565-3)). These template sequences arepreferably less than 50 nucleotides long, and may either be doublestranded, single stranded or partially single stranded DNA oligotemplates.

The oligonucleotide templates could either be synthetic DNA templates ortemplates generated as linearized plasmid DNA from a target-specificsequence cloned into a restriction site of a vector such as for examplea prokaryotic cloning vector (pUC13, pUC19) or PCR cloning systems suchas the TOPO cloning system of Invitrogen. The synthetic DNA templatesmay be produced according to techniques well known in the art. In apreferred embodiment of the present invention the oligonucleotidetemplates consist of partially single-stranded DNA oligo templatescomprising an RNA polymerase promoter sequence consisting of dsDNA. Inthis embodiment the target-specific short double stranded RNAs arefurther characterized by comprising at the 5′end nucleotides transcribedfrom the RNA polymerase promoter sequence and at the 3′end nucleotidescomplementary to the nucleotides transcribed from the promoter sequence.

A “Chain extending enzyme” as defined herein refers to an enzyme capableof forming an RNA polymer from ribonucleoside 5′triphosphates; the RNAformed is complementary to the DNA template. The enzyme addsmononucleotide units to the 3′-hydroxyl ends of the RNA chain and thusbuilds RNA in the 5′−>3′ direction, antiparallel to the DNA strand usedas template. Such chain extending enzymes could for example be DNAdependent polymerases such as DNA polymerase I, II and III; RNA-directedDNA polymerases such as RSV and AMV-polymerases; DNA-directed RNApolymerases such as E.coli RNA polymerase; RNA-directed RNA polymerasessuch as the bacteriophage RNA polymerases, also known as RNA replicases;or the bacterial polynucleotide phosphorylases.

In a preferred embodiment the chain extending enzyme is an RNApolymerase. Said RNA polymerases require the presence of a specificinitiation site within the DNA template. This initiation site, hereinafter referred to as “RNA polymerase promoter sequence”, is the sitewhere the RNA polymerase binds to the DNA template. It is also the siterecognized by the RNA polymerase as an initiation signal, to indicatewhere transcription to form RNA begins.

Accordingly, the present invention provides oligonucleotide templatescomprising an RNA polymerase promoter sequence consisting of dsDNAwherein the polymerase promoter sequence is recognized by an RNApolymerase. The term “recognized” as used herein intends to include alltruncated RNA polymerase promoter sequences shortened by one or morenucleotides at one or either side of the promoter sequence with no orlittle effect on the binding of the RNA polymerase to the initiationsite and with no or little effect on the transcription reaction. Forexample, Milligan et al. (Milligan F. et al., 1987, Nature (15)8783-8798) demonstrated for the T7 RNA polymerase that its promoter doesnot appear to require the DNA in the non-template strand in the region−17 to −14 and −3 to +6, since removing these nucleotides has littleeffect on the transcription reaction. Also, truncation of the templatestrand beyond position +2, i.e. positions +3 to +6, has little effect onthe yield of the reaction (Milligan F. et al., 1987, Nature (15)8783-8798). The thus obtained truncated RNA polymerase promotersequences are meant to be included as “RNA polymerase promoter sequencesrecognized by said RNA polymerase”. Thus, in a specific embodiment ofthe present invention the RNA polymerase promoter sequence consists ofthe truncated RNA polymerase promoter sequence wherein one or morenucleotides are deleted at one or either side of the template strand ofthe promoter sequence. Preferably, the truncated RNA polymerase promoterconsists of the T7 RNA polymerase promoter sequence truncated atpositions +3 to +6 at the 5′end of the template strand as shown in FIG.1.

Accordingly, it is a first object of the present invention to provide amethod for the synthesis of short double stranded target-specific RNAs.The method comprising the steps of a) combining a target-specific senseoligonucleotide template and a chain extending enzyme in a reactionmixture such that the template extended sense oligoribonucleotideproduct is formed; b) combining a target-specific antisenseoligonucleotide template and a chain extending enzyme in a reactionmixture such that the template extended antisense oligoribonucleotideproduct is formed; c) hybridizing the sense oligoribonucleotide productobtained in step a) with the antisense oligoribonucleotide productobtained in step b).

The chain-extending enzyme according to the method of the invention ispreferably an RNA polymerase selected from the group consisting of T7RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase. In a morepreferred embodiment the RNA polymerase consists of T7 RNA polymerase.

Accordingly, the oligonucleotide templates used in a method according tothe invention, comprise an RNA polymerase promoter sequence consistingof dsDNA, wherein the RNA polymerase promoter sequence is recognized byan RNA polymerase selected from the group consisting of T7 RNApolymerase, T3 RNA polymerase and SP6 RNA polymerase. In a preferredembodiment the RNA polymerase promoter sequence is recognized by T7 RNApolymerase. In a more preferred embodiment the T7 RNA polymerasepromoter sequence consists of the truncated T7 RNA polymerase promotersequence as shown in FIG. 1.

In a further embodiment, the oligonucleotide templates used in a methodaccording to the invention are characterized by being partially doublestranded DNA oligo templates comprising a double stranded RNA polymerasepromoter sequence which is extended at the 5′end of the template strandwith the target-specific template sequence, optionally extended with 2or 3 additional nucleotides. In a more preferred embodiment thetarget-specific template sequence comprises at the 5′end nucleotidestranscribed from the promoter sequence and at the 3′end nucleotidescomplementary to the nucleotides from the promoter sequence. In thespecific embodiment where the oligonucleotide templates comprise thetruncated T7 RNA polymerase promoter sequence shown if FIG. 1, thetarget-specific template sequence comprises at the 5′end two guanosine(g) nucleotides and at the 3′end two cytosine (c) nucleotides.

Accordingly, it is a second embodiment of the present invention toprovide a method for the synthesis of small interfering RNAs (siRNAs)comprising the steps of a) combining a sense siRNA template with a chainextending enzyme in a reaction mixture such that the template extendedsense oligoribonucleotide product is formed; b) combining an antisensesiRNA template with a chain extending enzyme in a reaction mixture suchthat the template extended antisense oligoribonucleotide product isformed; and c) hybridizing the sense oligoribonucleotide productobtained in step a) with the antisense oligoribonucleotide productobtained in step b); whereby the siRNA templates of step a) and b)comprise a double stranded RNA polymerase promoter sequence extended atthe 5′-end of the template strand with the target-specific templatesequence and 2 or 3 additional nucleotides. In a preferred embodimentthe chain-extending enzyme used in the synthesis of siRNAs consists ofan RNA polymerase, preferably selected from T7 RNA polymerase, T3 RNApolymerase or SP6 RNA polymerase. Accordingly the siRNA templates usedin the method according to the invention comprise an RNA polymerasepromoter sequence, which is recognized by an RNA polymerase, selectedfrom the group consisting of T7 RNA polymerase, T3 RNA polymerase andSP6 RNA polymerase. In a more preferred embodiment the chain-extendingenzyme is T7 RNA polymerase. Accordingly, in a preferred embodiment theRNA polymerase promoter sequence of the siRNA templates is recognized byT7 RNA polymerase. In a further embodiment the siRNA template comprisesthe double stranded truncated T7 RNA polymerase promoter sequence asshown in FIG. 1, wherein said truncated T7 RNA polymerase promotersequence is extended at the 5′end of the template strand according tothe method of the invention and wherein the target-specific templatesequence comprises at the 5′end the nucleotides transcribed from thepromoter sequence and at the 3′end nucleotides complementary to thenucleotides transcribed from the promoter sequence. In a specificembodiment the siRNA templates used in a method of the invention,comprise the double stranded truncated T7 RNA polymerase promotersequence as shown in FIG. 1, wherein said truncated T7 RNA polymerasepromoter sequence is extended at the 5′end of the template strandaccording to the method of the invention and wherein the target-specifictemplate sequence comprises at the 5′end two guanosine (g) nucleotidesand at the 3′end two cytosine (c) nucleotides.

The reaction conditions in either of the aforementioned methods toobtain a template extended oligoribonucleotide product are generallyknown in the art. In essence, the starting materials for enzymatictranscription to produce RNA are a DNA template, an RNA polymeraseenzyme and the nucleoside triphosphates (NTPs) for the four requiredribonucleotide bases, adenine, cytosine, guanine and uracyl, in areaction buffer optimal for the RNA polymerase enzyme activity. Forexample, the reaction mixture for an in vitro transcription using T7 RNApolymerase typically contains, T7 RNA polymerase (0.05 mg/ml),oligonucleotide templates (1 μM), each NTP (4 mM), and MgCl₂ (25 mM),which supplies Mg²⁺, a co-factor for the polymerase. This mixture isincubated at 37° C. and pH 8.1 (in for example 10 mM Tris-HCl buffer)for several hours (Milligan J. & Uhlenbeck O., 1989, Methods Enzymol(180) 51-62). Kits comprising the aforementioned components arecommercially available such as the MEGA shortscript™ T7 kit (Ambion).

Purification protocols to obtain the oligoribonucleotide products fromeither of the above mentioned methods are generally known in the art andcomprise amongst others gel electrophoresis, size exclusionchromatography, capillary electrophoresis and HPLC. Gel electrophoreseis typically used to purify the full-length transcripts from thereaction mixture, but this technique is not amendable to production atlarger scale. In a preferred embodiment of the present invention thepurification means to obtain the oligoribonucleotide products consistsof size exclusion chromatography, such as Sephadex G-25 resin,optionally combined with a phenol:chloroform:isoamyl extraction andethanol precipitation.

It is a third object of the present invention to provide kits to performthe methods according to the invention. In one embodiment the kitcomprises one or more of the following components a) instructions todesign target-specific sense and antisense oligonucleotide templates; b)a chain extending enzyme; c) transcriptionbuffers; d) the nucleosidetriphosphates (NTPs) for the four required ribonucleotide bases; e)purification means to obtain the sense and antisense oligoribonucleotideproducts. In a preferred embodiment of the present invention thechain-extending enzyme provided in the kit consists of an RNApolymerase, preferably an RNA polymerase selected from the groupconsisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNApolymerase. Even more preferably the chain extending enzyme provided ina kit according to the invention consist of T7 RNA polymerase.

The separating means provided in a kit according to the inventiongenerally refers to purification protocols known in the art to obtainoligoribonucleotide products from a reaction mixture and compriseamongst others gel electrophoresis, size exclusion chromatography,capillary electrophoresis and HPLC. In a preferred embodiment of thepresent invention the purification means provided in a kit according tothe invention consists of size exclusion chromatography columns orresins, such as Sephadex G-25 resin.

The instructions to design target-specific sense and antisenseoligonucleotide templates should contemplate the method exemplified inFIG. 1 of the present invention. In essence the method comprises thefollowing steps;

1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-xx(n₁₂₋₃₀)yy-3′. Wherein, x refers to the nucleotides transcribedfrom the promoter, y refers to the nucleotides complementary to thenucleotides transcribed form the promoter sequence, and n₁₂₋₃₀ refers toany oligonucleotide of 12 to 30 nucleotides

2) design a sense oligonucleotide template comprising the doublestranded RNA polymerase promoter sequence according to the inventionextended at the 5′end of the template strand with the complementoligonucleotide sequence of the target-specific sequence located in step1), optionally extended with two additional nucleotides.

3) design an antisense oligonucleotide template comprising the doublestranded RNA polymerase promoter sequence according to the inventionextended at the 5′end of the template strand with the reverseoligonucleotide sequence of the target-specific sequence located in step1), optionally extended with two additional nucleotides.

In a preferred embodiment the methods of the present invention use T7RNA polymerase as chain extending enzyme. In said embodiment the methodto design target-specific sense and antisense oligonucleotide templateswould comprise the following steps;

1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-gg(n₁₂₋₃₀)cc-3′ (SEQ ID NO: 47);

2) design a sense oligonucleotide template having the following sequence5′ TAATACGACTCACTATAGG (SEQ ID NO: 48)

3′ ATTATGCTGAGTGATATcc (n complement)₁₂₋₃₀ gg (SEQ ID NO: 49)—optionallyextended with two additional nucleotides, wherein (n complement)₁₂₋₃₀refers to the complement oligonucleotide sequence of the target-specificsequence located in step 1); and

3) design an antisense oligonucleotide template having the followingsequence 5′ TAATACGACTCACTATAGG (SEQ ID NO: 48) 3′ ATTATGCTGAGTGATATcc(n reverse)₁₂₋₃₀ gg (SEQ ID NO: 50)—optionally extended with twoadditional nucleotides, wherein (n reverse)₁₂₋₃₀ refers to the reverseoligonucleotide sequence of the target-specific sequence located in step1).

In a specific embodiment the methods of the present invention are usedfor the synthesis of small interfering RNAs (siRNAs). In said embodimentthe method to design target-specific sense and antisense siRNA templateswould comprise the following steps;

1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-xx(n₁₅₋₃₀)yy-3′. Wherein, x refers to the nucleotides transcribedfrom the promoter, y refers to the nucleotides complementary to thenucleotides transcribed form the promoter sequence, and n₁₅₋₃₀ refers toany oligonucleotide of 15 to 30 nucleotides;

2) design a sense oligonucleotide siRNA template comprising the doublestranded RNA polymerase promoter sequence according to the inventionextended at the 5′end of the template strand with the complementoligonucleotide sequence of the target-specific sequence located in step1), extended with two additional nucleotides, preferably two adenineresidues;

3) design an antisense oligonucleotide siRNA template comprising thedouble stranded RNA polymerase promoter sequence according to theinvention extended at the 5′end of the template strand with the reverseoligonucleotide sequence of the target-specific sequence located in step1), extended with two additional nucleotides, preferably two adenineresidues.

In the specific embodiment, where the methods to synthesize siRNAs makeuse of T7 RNA polymerase as chain extending enzyme, the method to designtarget-specific sense and antisense siRNA templates would comprise thefollowing steps;

1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-gg(n₁₅₋₃₀)cc-3′ SEQ ID NO: 51);

2) design a sense oligonucleotide siRNA template having the followingsequence 5′ TAATACGACTCACTATAGG (SEQ ID NO: 52)

3′ ATTATGCTGAGTGATATcc (n complement)₁₅₋₃₀ gg aa (SEQ ID NO: 53)

wherein (n complement)₁₅₋₃₀ refers to the complement oligonucleotidesequence of the target-specific sequence located in step 1); and

3) design an antisense oligonucleotide siRNA template having thefollowing sequence 5′ TAATACGACTCACTATAGG (SEQ ID NO: 52)

3′ ATTATGCTGAGTGATATcc (n reverse)₁₅₋₃₀ gg aa (SEQ ID NO: 54)

wherein (n reverse)₁₅₋₃₀ refers to the reverse oligonucleotide sequenceof the target-specific sequence located in step 1).

Accordingly, the present invention provides a kit for the synthesis ofshort double stranded target-specific RNAs the kit comprising at leastone of the following components; a) instructions to designtarget-specific sense and antisense oligonucleotide templates; b) achain extending enzyme; c) transcriptionbuffers; d) the nucleosidetriphosphates (NTPs) for the four required ribonucleotide bases; e)purification means to obtain the sense and antisense oligoribonucleotideproducts.

Thus in a further embodiment the present invention provides kits for thesynthesis of small interfering RNAs the kit comprising at least one ofthe following components; a) instructions to design target-specificsense and antisense siRNA templates; b) a chain extending enzyme; c)transcriptionbuffers; d) the nucleoside triphosphates (NTPs) for thefour required ribonucleotide bases; e) purification means to obtain thesense and antisense oligoribonucleotide products.

It is also an object of the present invention to provide the means forany of the disclosed methods for the in vitro synthesis of short doublestranded RNAs. Accordingly the present invention provides;

-   -   i) a method to design target-specific sense and antisense        oligonucleotide templates    -   ii) a chain extending enzyme according to the invention for use        in a method for the in vitro synthesis of short double stranded        RNAs    -   iii) purification means to obtain the sense and antisense        oligoribonucleotide products.    -   iv) reagents for the reaction mixture such that the sense and        antisense oligoribonucleotide products are formed from the        target-specific sense and antisense oligonucleotide templates        using a chain extending enzyme according to the invention

It is a further object of the present invention to use the siRNAsobtainable by a method of the present invention in a process forinhibiting expression of a target gene in a cell. The process comprisingintroduction of siRNAs obtainable by a method of the present invention,into a cell.

The target gene may be a gene derived from the cell (i.e., a cellulargene), an endogenous gene (i.e., a cellular gene present in the genome),a transgene (i.e., a gene construct inserted at an ectopic site in thegenome of the cell), or a gene from a pathogen which is capable ofinfecting an organism from which the cell is derived. Depending on theparticular target gene and the dose of double stranded RNA materialdelivered, this process may provide partial or complete loss of functionfor the target gene.

The cell with the target gene may be derived from or contained in anyorganism. The organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. The cell having the target gene maybe from the germ line or somatic, totipotent or pluripotent, dividing ornon-dividing, parenchyma or epithelium, immortalized or trans- formed,or the like. The cell may be a stem cell or a differentiated cell. Celltypes that are differentiated include adipocytes, fibroblasts, myocytes,cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes,lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine orexocrine glands.

The isolated RNA obtainable by a method of the present inventionconsists of target-specific short double stranded RNAs, wherein saidtarget-specific short double stranded RNAs are less than 50 nucleotides,preferably less than 30 nucleotides long, even more preferably 30-12nucleotides, characterized by comprising at the 5′end nucleotidestranscribed from the promoter sequence and at the 3′end nucleotidescomplementary to the nucleotides transcribed from the promoter sequence,preferably the number of nucleotides transcribed from the promotersequence and the number of nucleotides complementary to the nucleotidestranscribed from the promoter sequence consist of 2, 3, or 4nucleotides, more preferably of 2 nucleotides.

The short double stranded RNAs obtainable by a method of the presentinvention are optionally extended at the 3′end with 2 or 3 additionalnucleotides and could in a further embodiment of the present inventionbeing represented as having the following sense sequence5′-xx(n₁₂₋₃₀)yy-3′ wherein x refers to the nucleotides transcribed fromthe promoter sequence, y refers to the nucleotides complementary to thenucleotides transcribed form the promoter sequence, and n₁₂₋₃₀ refers toany oligonucleotide of 12 to 30 nucleotides. In a specific embodimentthe short double stranded RNAs have as sense sequence 5′-gg(n₁₅₋₃₀)cc-3′(SEQ ID NO: 51) wherein g refers to the nucleotide guanosine transcribedfrom the truncated T7 RNA polymerase promoter sequence (as shown in FIG.1), c refers to the nucleotide cytosine complementary to the nucleotidestranscribed form the truncated T7 RNA polymerase promoter (as shown inFIG. 1) sequence, and n₁₅₋₃₀ refers to any oligonucleotide of 15 to 30nucleotides.

The RNA may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining the RNA. Methods for oral introduction include direct mixingof the RNA with food of the organism, as well as engineered approachesin which a species that is used as food is engineered to express theRNA, then fed to the organism to be affected. For example, the RNA maybe sprayed onto a plant or a plant may be genetically engineered toexpress the RNA in an amount sufficient to kill some or all of apathogen known to infect the plant.

Physical methods of introducing nucleic acids, for example, injectiondirectly into the cell or extracellular injection into the organism, mayalso be used. We disclose herein that in HeLa cells, double-stranded RNAintroduced outside the cell inhibits gene expression.

Vascular or extravascular circulation, the blood or lymph system, thephloem, the roots, and the cerebrospinal fluid are sites where the RNAmay be introduced. A transgenic organism that expresses RNA from arecombinant construct may be produced by introducing the construct intoa zygote, an embryonic stem cell, or another multipotent cell derivedfrom the appropriate organism.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or otherwise increase inhibition of thetarget gene.

The present invention may be used to introduce RNA into a cell for thetreatment or prevention of disease. For example, dsRNA may be introducedinto a cancerous cell or tumor and thereby inhibit gene expression of agene required for maintenance of the carcinogenic/tumorigenic phenotype.To prevent a disease or other pathology, a target gene may be selectedwhich is required for initiation or maintenance of thedisease/pathology. Accordingly, in a further embodiment the inventionprovides a pharmaceutical composition comprising short double strandedRNAs obtainable by a method of the present invention to inhibit geneexpression of a target gene and an appropriate carrier. The compositionmay be administered in any suitable way, e.g. by injection, by oral,intra-ocular, topical, nasal, rectal application etc. The carrier may byany suitable pharmaceutical carrier, preferably, a carrier is used,which is capable of increasing the efficacy of the RNA molecules toenter the target cells, for example liposomes, natural viral capsids orby chemically or enzymatically produced artificial capsids or structuresderived therefrom.

Another utility of the present invention could be a method ofidentifying gene function in an organism comprising the use ofdouble-stranded RNA to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics would envision determining the function of uncharacterizedgenes by employing the invention to reduce the amount and/or alter thetiming of target gene activity. The invention could be used indetermining potential targets for pharmaceutics, understanding normaland pathological events associated with development, determiningsignaling pathways responsible for postnatal development/aging, and thelike. The increasing speed of acquiring nucleo-tide sequence informationfrom genomic and expressed gene sources, including total sequences forthe human, mouse, yeast, D. melanogaster, and C. elegans genomes, can becoupled with the invention to determine gene function in an organism(e.g., nematode). The preference of different organisms to useparticular codons, searching sequence databases for related geneproducts, correlating the linkage map of genetic traits with thephysical map from which the nucleotide sequences are derived, andartificial intelligence methods may be used to define putative openreading frames from the nucleotide sequences acquired in such sequencingprojects.

A simple assay would be to inhibit gene expression according to thepartial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product.

It is thus an object of the present invention to provide a method toinhibit expression of a target gene in a cell comprising introduction ofRNA into a cell wherein said RNA comprises target-specific short doublestranded RNA, wherein said target-specific short double stranded RNA isless than 50 nucleotides, preferably less than 30 nucleotides long, evenmore preferably 30-12 nucleotides long, characterized by comprising atthe 5′end nucleotides transcribed from the promoter sequence and at the3′end nucleotides complementary to the nucleotides transcribed from thepromoter sequence wherein said promoter sequence is being recognized byan RNA polymerase. In a further embodiment the promoter sequence isbeing recognized by an RNA polymerase selected from the group consistingof T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase.

This invention will be better understood by reference to theExperimental Details that follow, but those skilled in the art willreadily appreciate that these are only illustrative of the invention asdescribed more fully in the claims that follow thereafter. Additionally,throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

EXAMPLE 1 EGFP and GL3 Specific short dsRNAs Transcribed in Vitro,induce RNA Interference in Human Cells

Materials and Methods

Plasmid Constructs

Luciferase+ was expressed from the plasmid pGL3-control (Promega). EGFPwas expressed from EGFP/pcDNA5-FRT, which contains the EGFP gene frompEGFP (Clontech) directionally ligated into the HindIII and NotI sitesof the mammalian expression vector pcDNA5/FRT (Invitrogen).

In Vitro Transcription and Hybridization of siRNAs

Oligo template strands were hybridized to a sense T7 promoter sequence(5′TAATACGACTCACTATAGG) (SEQ ID NO: 55) in 10 mM Tris-HCl pH 9.0, 100 mMNaCl, 1 mM EDTA by boiling for 2′ and cooling slowly to room temperatureover 2-3 hr. Transcription was performed using the MEGAshortscript™ T7kit (Ambion) according to the manufacturer's instructions. siRNA strandswere purified over G-25 spin columns, phenol:chloroform:isoamyl alcohol(25:24:1) extracted using Heavy Phase-Lock Gels (Eppendorf), and ethanolprecipitated overnight at −80° C. Complementary siRNA strands werehybridized in 1 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0 by boiling for 2′and cooling slowly to room temperature over 2-3 hr. Hybridization wasassessed by running the ds- and ss-siRNAs on non-denaturing 20%polyacrylamide TBE gels.

Cell Lines and Transfection

HeLa cells were grown in DMEM with high glucose and 1-glutamine(Invitrogen) supplemented with 1.8 mM 1-glutamine, 9% FBS, and 45 U/lpen/strep. Cells were transfected in a manner similar to that describedin Elbashir et al. (2001). 24 hr before transfection, cells weretrypsinized and diluted with growth medium lacking antibiotics to 3×10⁵cells/ml. 0.5 ml of cells were seeded into each well of a 24-well plate.The cells were transfected with 1 μg of GL3-control or EGFP/pcDNA5-FRTreporter constructs and 50 pmol of single-stranded or 25 pmoldouble-stranded siRNAs, except where otherwise noted, usingLipofectamine™ 2000 (LF2000; Invitrogen) according to the manufacturer'sinstructions. Specifically, we used 21 μl of LF2000 per well in 48 μl ofserum-free medium lacking antibiotics. The diluted LF2000 waspre-incubated at room temperature for 1′ prior to mixing with reporterand/or siRNAs diluted in the same medium to 50 μl total volume.Complexes were then incubated at room temperature for 20′ before beingadded to the cells. EGFP and GL3 reporter gene assays were performedafter 24 hrs. For the JNK2α1 siRNA experiment and GL3 siRNA doseresponse experiment, 6-well plates were used. Cell numbers wereincreased 4-fold and reagent amounts 5-fold. For the JNK2α1 siRNAexperiment, cells were harvested for RNA isolation and proteinextraction were approximately 48 hr post-transfection.

Reporter Gene Assays

FACS analysis of EGFP-transfected cells was performed using a FACScan(Beckton-Dickinson). Cells were trypsinized and washed with PBS prior toresuspension in FACS fixing solution (PBS+1% formaldehyde). Transfectionefficiencies were estimated by comparing samples transfected with waterwith those transfected with EGFP/pcDNA5-FRT and were typically 75-90%.The extent of RNAi induced by transcribed or synthetic siRNAs wasestimated from the change in the mean GFP fluorescence in samples withor without cotransfected siRNAs.

For luciferase assays, cells were trypsinized and 100 μl aliquots weretransferred to triplicate wells in white 96-well tissue culture plates.Assays were performed using the Luc-Screen™ System (Applied Biosystems)and a TopCount-NXT™ Microplate Scintillation and Luminescence Counter(Packard) according to the manufacturers' instructions.

Northern and Western Blotting

Total RNA was prepared from samples of approximately 10⁶ HeLa cellsusing the RNeasy Mini Kit (Qiagen) according to the manufacturer'sinstructions. Samples were run on pre-cast MOPS Latitude RNA agarosegels (BioWhittaker Molecular Applications) and transferred to Hybond-XLnylon membranes (AP Biotech) according to the manufacturers'instructions. DNA probes were made using the Rediprime II system (APBiotech) according to the manufacturer's instructions. Hybridization wasperformed in Rapid-Hyb solution (AP Biotech) according to themanufacturer's instructions.

Nuclear and cytoplasmic protein extracts were made by the methoddescribed in Gordon (1991), substituting Complete Protease InhibitorCocktail (Roche) for leupeptin, aprotinin, and pepstatin and omittingsodium deoxycholate.

Extracts were run on 4-20% SDS polyacrylamide minigels (Invitrogen) withRainbow protein marker (AP Biotech) and electroblotted onto 0.2 μmTransblot nitrocellulose membranes (BioRad). The blots were rinsed inPBS+0.05% Tween-20 and incubated overnight at 4° C. with 1:150 dilutedJNK2 D2 mouse monoclonal antibody (Santa Cruz Biotech) in PBS/Tween-20with 5% milk powder. The blots were then washed three times inPBS/Tween-20 before a 45 min incubation with 1:3000 HRP-conjugated goatanti-mouse antibodies (BioRad). Three more washes and a 30 minincubation in PBS/Tween-20 were performed before detection by the ECLsystem (AP Biotech).

Results

RNAi has been previously demonstrated using siRNAs that aredouble-stranded except for two 3′ overhanging nucleotides (Elbashir etal. 2001; Caplen et al. 2001). In order to relatively quickly andinexpensively create a variety of siRNAs for multiple cellular targets,we designed a scheme to generate the molecules using in vitrotranscription techniques.

Milligan et al. (1987) describe the use of partially single-stranded DNAoligo templates for transcription by T7 DNA polymerase. The standard T7minimal promoter includes three guanosine nucleotides at the 3′ endwhich are incorporated as the first three bases of the transcript.However, the third guanosine can usually be replaced with othernucleotides without significant reduction of in vitro transcriptionyields (Milligan et al. 1987). Therefore, siRNAs produced by this methodshould include two 5′ guanosine nucleotides. Two complementary cytosinenucleotides are needed near the 3′ end of each siRNA strand to base pairwith the 5′ guanosines on the other strand. siRNAs of a given lengthproduced in this manner should be able to target sequences appearingapproximate once every 250 nucleotides on average in an mRNA.

We designed DNA oligo templates with the constraints above in mind (FIG.1). Each template is used to transcribe one strand of an siRNA. Thestrands are crudely purified by passage over a Sephadex G-25 sizeexclusion column, phenol:chloroform extraction, and ethanolprecipitation. The strands are then resuspended in annealing buffer andhybridized by boiling and slow cooling.

Our in vitro transcribed siRNAs differ from the chemically synthesizedvariety used previously in two ways. First, all other reported siRNAshave been highly purified. Second, in vitro transcribed siRNAs retain a5′ triphosphate group. Like the siRNA species produced in vivo as partof the natural RNAi mechanism, chemically synthesized RNAoligonucleotides used to make siRNAs have carried 5′ monophosphates. Inorder to determine if these differences render our siRNAs incompetent toinduce RNAi, we first tested double-stranded siRNAs designed to targettwo reporter genes—EGFP and GL3 (FIGS. 2 and 3). Mean results andstandard errors from at least three independent experiments are shown(FIG. 2B, FIG. 3B).

The transcribed GL3 ds siRNA 1 reduced luciferase activity from thecotransfected pGL3-control reporter plasmid by approximately 5-foldwhile the antisense strand alone in double-molar concentration had noeffect. A similar result was observed with a chemically synthesized GL3ds siRNA (ds1 RNA oligos). The second transcribed siRNA had a moremodest effect. While the third transcribed siRNA had a strong effect,significant activity was also seen from the antisense strand alone. Thestrength of the effect from the double-stranded species is dosedependent (FIG. 2C) and modifies steady-state RNA levels. EGFP ds siRNA2 also had a modest effect on luciferase activity (FIG. 2B). However,this effect appears to be non-specific and shows a limited response atincreasing doses.

The same transcribed EGFP ds siRNA 2 strongly reduced GFP fluorescencein cells cotransfected with the EGFP/pcDNA5-FRT reporter (FIG. 3B). Muchmore modest effects were evident from sense or antisense strands aloneor from a chemically synthesized siRNA (EGFP ds1 RNA oligos) or itscomponent parts. GFP fluorescence was not affected by the non-specificGL3 ds siRNA 3.

The above mentioned in vitro transcribed (IVT) luciferase ds siRNAsyielded inhibition of luciferase activity to a different extent. As apositive control for RNAi activity, we used a chemically synthesizedluciferase ds siRNA. In our hands, luciferase activity from thecotransfected pGL3-control reporter plasmid was reduced approximately5-fold by the synthetic ds siRNAs. Transfection efficiencies for allexperiments varied between 91 and 95%. One IVT-luciferase ds siRNA (GL3ds siRNA 1) reduced luciferase activity 78%, while the antisense RNAstrand alone at twice the molar concentration of the ds siRNA had noeffect. The second IVT-siRNA (GL3 ds siRNA 2) we tested had a moremodest effect (36% inhibition). While the third IVT-siRNA had a strongeffect (82% inhibition), significant activity was also seen from theantisense RNA strand alone (55% inhibition), confounding the result. Amixture of the three IVT-siRNAs, each at one-third the molarconcentration used for them individually, had an intermediate effect(70% inhibition) rather than a synergistic one, suggesting that theremay be no advantage to using multiple siRNAs to target the same gene. Wecould show the inhibitory effect from the ds species to bedose-dependent. While a non-specific GFP siRNA (GFP ds siRNA 2) also hada modest effect (46% inhibition) on luciferase activity, this appears tobe non-specific and shows a limited response at increasing doses (datanot shown).

The same IVT-GFP ds siRNA (GFP ds siRNA 2) strongly reduced GFPfluorescence in cells cotransfected with the GFP/pcDNA5-FRT reporter(87% inhibition). Much more modest effects were evident from sense (41%)or antisense (19%) strands alone or from a chemically synthesized siRNA(GFP ds1 RNA oligo) or its component parts. GFP fluorescence was, asexpected, not affected by the non-specific luciferase ds siRNA 3.

To demonstrate that endogenous gene expression can also be affected bytranscribed siRNAs, we targeted the products of the JNK2α1 (FIG. 4A) andCDS-1 (FIG. 4B) genes. Western and Northern blot analysis revealedspecific reduction of JNK2α1 protein and RNA levels in samples innuclear extracts of HeLa cells transfected with either a transcribedsiRNA (JNK2α1 ds siRNA 1—estimated 87% reduction) or a chemicallysynthesized siRNA (JNK2α1 ds1 RNA oligos—estimated 76% reduction) whencompared to cells transfected with water (mock), EGFP/pcDNA5-FRT plasmidas a transfection control (EGFP DNA only), single strands of siRNAs, ora non-specific siRNA (EGFP ds siRNA 2).

Western blot analysis revealed modest (up to 67%) reduction of CDS 1protein levels in cytoplasmatic extracts of HeLa cells transfected withCDS 1-specific IVT-siRNAs (FIG. 4) but not in cells transfected with anunspecific siRNA when compared to mock-transfected cells.

EXAMPLE 2 Mouse Insr Specific short dsRNAs Transcribed in Vitro,Knockdown Insr in Liver of Balb/C Mice

Male Balb/C mice (approx 25 g) (standard housing, free access tochow/water) received a tail vein injection of either saline, 2.3 ml, orsaline containing 40 micrograms of siRNA directed against the murineinsulin receptor (NCBI accession number NM_(—)010568; bases 2536-2556)prepared,by the truncated T7 promoter method of in vitro transcription,along with 800 U RNase inhibitor.

The injections were administered as rapidly as possible (8-10 seconds).Two control and two siRNA treated mice were sacrificed at 24, at 48 andat 72 hours; the liver was quickly removed,weighed, and frozen in dryice/isopropanol. Total RNA was extracted using pulverized frozen tissueand RNEasy Maxi kits (Qiagen).

After first strand cDNA synthesis, mRNA for the insulin receptor wasassayed by Q-PCR using the Smart Cycler (primers: F 3526-3548, R3744-3768) and results were normalized to cyclophilin A expression alsoby Q-PCR (bases 157-182 and 496-521 of NCBI accession numberNM_(—)017101).

1. A method for the synthesis of target-specific short double strandedRNAs of less than 30 nucleotide long comprising the steps of: a)combining a target-specific sense oligonucleotide template and a T7 RNApolymerase in a reaction mixture such that a template extended, senseoligoribonucleotide, product is formed; b) combining a target-specificantisense oligonucleotide template and T7 RNA polymerase in a reactionmixture such that a template extended, antisense oligoribonucleotide,product is formed; and c) hybridizing the sense oligoribonucleotideproduct obtained in step a) with the complementary antisenseoligoribonucleotide product obtained in step b), characterized in that;the oligonucleotide templates of step a) and b) comprise an RNApolymerase promoter sequence consisting of the truncated T7 RNApolymerase promoter sequence as set forth in SEQ ID NO: 56, extended atthe 5′-end of the template strand with the target-specific templatesequence, wherein said target-specific template sequence comprises atthe 5′-end two guanosine (g) nucleotides and at the 3′-end two cytosine(c) nucleotides, wherein said two cytosine nucleotides being the firsttwo nucleotides of said promoter sequence.
 2. A method for the synthesisof target-specific short double stranded RNAs of less than 30 nucleotidelong comprising the steps of: a) combining a target-specific senseoligonucleotide template and a T7 RNA polymerase in a reaction mixturesuch that a template extended, sense oligoribonucleotide, product isformed; b) combining a target-specific antisense oligonucleotidetemplate and T7 RNA polymerase in a reaction mixture such that atemplate extended, antisense oligoribonucleotide, product is formed; andc) hybridizing the sense oligoribonucleotide product obtained in step a)with the complementary antisense oligoribonucleotide product obtained instep b), characterized in that; wherein the oligonucleotide templates ofstep a) and b) are characterized by being partially double stranded DNAoligo templates comprising a double stranded RNA polymerase promotersequence consisting of the truncated T7 RNA polymerase promoter sequenceas set forth in SEQ ID NO: 56, extended at the 5′-end of the templatestrand with the target-specific template sequence, wherein saidtarget-specific template sequence comprises at the 5′-end two guanosine(g) nucleotides and at the 3′-end two cytosine (c) nucleotides, whereinsaid two cytosine nucleotides being the first two nucleotides of saidpromoter sequence.
 3. A method for the synthesis of small interferingRNAs (siRNAs) of 12-30 nucleotides comprising the steps of; a) combininga sense siRNA template with T7 RNA polymerase in a reaction mixture suchthat a template extended sense oligoribonucleotide product is formed; b)combining an antisense siRNA template with T7 RNA polymerase in areaction mixture such that a template extended antisenseoligoribonucleotide product is formed; and c) hybridizing the senseoligoribonucleotide product obtained in step a) with the antisenseoligoribonucleotide product obtained in step b); whereby the siRNAtemplates of step a) and b) comprise a double stranded RNA polymerasepromoter sequence consisting of the truncated T7 RNA polymerase sequenceas set forth in SEQ ID NO: 56, extended at the 5′-end of the templatestrand with the target-specific template sequence as defined in claim 1,wherein the template further contains 2 or 3 additional nucleotides.